Many researchers experience a trade-off between maintaining cell morphology and preserving gene expression information in the same tissue sample. Paraffin-embedded tissue samples generally show good morphology but gene expression data are severely compromised. Conversely, frozen tissue samples remain the gold standard for obtaining high quality gene expression information, but tissue morphology from frozen material is inferior compared to the morphology of paraffin-embedded tissue samples. Given the empirical foundation of microscopic analysis for current methods of tissue diagnosis and prognosis, the transfer of molecular methods into pathology practice has been greatly hampered. Indeed, the development of improved methods for tissue processing for transcript profiling of pathological samples are still necessary (Perlmutter M A, Best C J, Gillespie J W, Gathright Y, Gonzalez S, Velsaco A, Linehand W M, Emmert-Buck M R, Chuaqui R F: Comparison of snap freezing versus ethanol fixation for gene expression profiling of tissue specimens. J Mol Diagn 6(4):371-7, 2004). For gene expression analysis, the presence of intact and extractable nucleic acids from the test material is mandatory. Because the emerging role of transcript profiling studies in research and clinical work, a nucleic acid-friendly fixative with the morphological detail of formalin-fixed paraffin-embedded (FFPE) tissues should replace formalin as the primary (human) tissue fixative.
The cellular abundance of a particular RNA transcript is tightly regulated by the balance between its transcription and degradation rate. It is important for gene expression studies that the measured RNA population reflects the actual transcriptosome present at the time of tissue or cell collection as closely as possible. As RNA is rapidly degraded by ribonucleases, it is of paramount importance to reduce or halt endogenous enzyme activity as quickly as possible prior to or at the time of tissue or cell collection. One way of accomplishing this ‘status quo’ situation is by freezing techniques such as snap-freezing in liquid nitrogen. However, as known by those persons skilled in the art, the freezing process results in suboptimal microscopical detail of tissue architecture and cell morphology. Moreover transport of frozen tissues requires specialist shipment and is more costly and risky (chance of defrosting) than transporting paraffin-embedded material. For example, if gene expression studies are performed in a central reference laboratory, e.g. in clinical trials, frozen samples have to be properly prepared for courier shipment. Sometimes, international sample carriage is not possible.
Another problem in the art is the limited tissue biopsy availability. A single small-sized biopsy (e.g. needle biopsy) may not provide sufficient tissue for both classical histopathological analyses and molecular pathology assays. Consequently, it may be required to collect two tissue samples which causes additional distress and injury to the patient.
The extraction of high molecular weight DNA from paraffin-embedded tissue has been reported (e.g. Dubeau L, Chandler L A, Gralow J R, Nichols P W, Jones P A: Southern blot analysis of DNA extracted from formalin-fixed pathology specimens. Cancer Res 1986, 46:2964-2969; Greer C E, Peterson S L, Kiviat N B, Manos M M: PCR amplification from paraffin-embedded tissues. Effects of fixative and fixation time. Am J Clin Pathol 1991, 95:117-124; Inoue T, Nabeshima K, Kataoka H, Koono M: Feasibility of archival non-buffered formalin-fixed and paraffin-embedded tissues for PCR amplification: an analysis of resected gastric carcinoma. Pathol Int 1996, 46:997-1004; Ren Z P, Sällström J, Sundström C, Nistér M, Olsson Y: Recovering DNA and optimizing PCR conditions from microdissected formalin-fixed and paraffin-embedded materials. Pathobiology 2000, 68:215-217). However, prolonged exposure of tissues to the fixative formaldehyde results in irreversible cross-linking of proteins and nucleic acids, causing the maximum PCR amplicon size to be limited (Finkelstein S D, Sayegh R, Christensen S, Swalsky P A: Genotypic classification of colorectal adenocarcinoma. Cancer 1993, 71: 3827-3838). In addition extensive fixation in formaldehyde leads to nucleic acid scission, further diminishing the efficiency of PCR-based analysis and amplicon size. Although DNA survives fixation and embedding reasonably well, RNA content is seriously decreased due to the combination of the presence of RNase activity in virtually all tissues and the use of excessive heating during the infiltration and embedding procedures of the tissue processing. Indeed, it is more difficult to obtain high molecular weight RNA from (archival) paraffin-embedded material. Extraction of RNA with a maximal length of 600 base pairs has been described (Stanta G, Schneider C: RNA extracted from paraffin-embedded human tissues is amenable to analysis by PCR amplification. Biotechniques 1991, 11:304, 306, 308; Krafft A E, Duncan B W, Bijwaard K E, Taubenberger J K, Lichy J H: Optimization of the Isolation and Amplification of RNA From Formalin-fixed, Paraffin-embedded Tissue: The Armed Forces Institute of Pathology Experience and Literature Review. Mol Diagn 1997, 2:217-230; Goldsworthy S M, Stockton P S, Trempus C S, Foley J F, Maronpot R R: Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue. Mol Carcinog 1999, 25:86-91; Specht K, Richter T, Müller U, Walch A, Höfler M W: Quantitative gene expression analysis in microdissected archival tissue by real-time RT-PCR. J Mol Med 2000, 78:B27; Specht K, Richter T, Muller U, Walch A, Werner M, Hofler H: Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue. Am J Pathol 2001, 158:419-429; Paska C, Bogi K, Szilak L, Tokes A, Szabo E, Sziller I, Rigo J Jr, Sobel G, Szabo I, Kaposi-Novak P, Kiss A, Schaff Z: Effect of formaline, acetone, and RNAlater fixatives on tissue preservation and different size amplicons by real-time PCR from paraffin-embedded tissue. Diagn Mol Pathol 2004, 13:234-240)
However, such RNA fragment sizes severely limit the suitability of the RNA for certain molecular profiling applications such as the construction of full length cDNA libraries. Even if the average RNA fragment size would be sufficiently large to allow paraffin-embedded tissues or cells to be used for RT-PCR, nucleic acid amplification procedures and microarray analyses, the reliability and reproducibility of quantitative gene expression studies are questionable in the presence of degraded and chemically modified RNA, especially since the different mRNA species from the mRNA pool are most likely not affected to the same degree/extent.
A number of fixative formulations have been described in the art. U.S. Pat. No. 6,319,683 is based on controlling the reactivity of the fixating components by quenching the excess formaldehyde with a formaldehyde reactive agent. U.S. Pat. No. 5,976,829 describes a fixative comprising aldehyde, alcohol and CDTA. WO 03/029783 describes the protection of the tissue specimen by impregnation with an osmotically buffered amino acid solution prior to fixation with an acetone-based fixative, which would obviate the need for a cross-linking agent. WO 00/06780 discloses a method for maintaining RNA integrity in biological materials by means of an RNA preservation medium. Although the patented medium does keep the RNA intact (Mutter G L, Zahrieh D, Liu C, Neuberg D, Finkelstein D, Baker H E, Warrington J A: Comparison of frozen and RNALater solid tissue storage methods for use in RNA expression arrays. BMC Genomics 2004, 5:88), in histological applications a variable outcome on tissue morphology and immunostaining has been observed (Florell S R, Coffin C M, Holden J A, Zimmermann J W, Gerwels J W, Summers B K, Jones D A, Leachman S A: Preservation of RNA for functional genomic studies: a multidisciplinary tumor bank protocol. Mod Pathol 2001, 14:116-128; Roos-van Groningen M C, Eikmans M, Baelde H J, de Heer H J, Bruijn J A: Improvement of extraction and processing of RNA from renal biopsies. Kidney Int 2004, 65:97-105).
U.S. Pat. No. 6,379,921 described a method using a procedure based on a zinc-containing aqueous fixative, an acetone-based clearing agent and molten resin. However, the resulting tissue blocks must be sectioned and processed differently from routine paraffin blocks which may complicate the work-flow in a routine pathology lab.
For future tissue conservation of pathology specimens, it would be desirable to satisfy both histological and molecular biological needs. An uncomplicated fixation and paraffin embedding method that results in tissue sections with the same morphological characteristics as formalin-fixed paraffin-embedded (FFPE) tissues, while preserving nucleic acid integrity would have an important impact on the feasibility and logistics of clinical trials. In addition, such method would greatly facilitate the introduction of gene expression analyses in routine pathology laboratories, especially if it requires no or only limited modification of standard routine downstream pathology protocols.